ES2442951T3 - DNA sequence and preparation of major allergens of cereal group 4 - Google Patents

Abstract

DNA molecules (I), corresponding to the sequence of the major allergens of cereal pollen are new. (I) have sequences (1; 1603 nucleotides (nt)); (3; 1644 nt); (5; 1608 nt); (7; 1603 nt) or (9; 1603 nt), all reproduced. DNA molecules (I), corresponding to the sequence of the major allergens of cereal pollen comprise sequences comprising 1603 nucleotides (SEQ ID No.:1) (nt)); 1644 nucleotides (SEQ ID No.:3; ); 1608 nucleotides (SEQ ID No.:5; ); 1603 nucleotides (SEQ ID No.:7; ) or 1603 nucleotides (SEQ ID No.:9; ). Independent claims are also included for: (1) DNA (Ia) that hybridizes to (I) under stringent conditions and is derived from DNA of Poaceae; (2) DNA (Ib) that encodes a polypeptide that is immunologically cross-reactive with the major allergens Sec c 4 ( Secale cereale); Hor v 4 ( Hordeum vulgare) or Tri a 4 ( Triticum aestivum) and derived from Poaceae; (3) DNA fragment, or combination of fragments, of (I), (Ia) or (Ib) that encodes an immunomodulatory T-cell reactive fragment (X) of Group 4 Poaceae allergens; (4) a DNA corresponding to any of the new DNAs or fragments that encodes an (X) but is modified by targeted mutation of individual codons, deletion or addition; (5) recombinant DNA expression vector or cloning system containing any of the new DNAs linked to expression control sequences; (6) host organism transformed with any of the new DNAs or the vector of (5); (7) preparing a polypeptide (II) encoded by the new DNAs by culturing the organisms of (6); and (8) (II) having any of the sequences, comprising 518 amino acids (SEQ ID No.:2; 520 amino acids (SEQ ID No.:4; ); 518 amino acids (SEQ ID No.:6; ); 518 amino acids (SEQ ID No.:8) or 518 amino acids (SEQ ID No.: 10; ) or their mature forms. ACTIVITY : Antiallergic. No biological data given. MECHANISM OF ACTION : Vaccine.

Description

DNA sequence and preparation of major allergens of cereal group 4

Background of the invention

The present invention relates to the preparation of DNA sequences of major allergens of group 4 of

5 cereals (Triticeae). The invention also includes fragments, novel combinations of partial sequences and point mutations with hypoallergenic effect. Recombinant DNA molecules and derived polypeptides, fragments, new combinations of partial sequences and variants can be used for the therapy of pollen allergy diseases. The prepared recombinant proteins can be used for in vitro and in vivo diagnosis of pollen allergies.

10 Type 1 allergies are important worldwide. Up to 20% of the population of industrialized countries suffer from ailments such as allergic rhinitis, conjunctivitis or bronchial asthma. These allergies are caused by allergens present in the air (aeroallergens), which are released by sources from different sources, such as pollen from plants, mites, cats or dogs. Up to 40% of these type 1 allergies also show specific IgE reactivity with grass pollen allergens, among others, pollen allergens

15 cereals (Freidhoff et al., 1986, J. Allergy Clin. Immunol. 78, 1190-2001). Among the allergens of cereal pollen, rye allergens are especially important.

The triggers of type 1 allergies are proteins, glycoproteins or polypeptides. In sensitized people, after absorption in the mucous membranes, these allergens react with the IgE antibodies that are attached to the surface of the mast cells. When two IgE molecules bind through an allergen they

20 results in the release of mediators (eg, histamine, prostaglandins) and cytokines through effector cells and thereby trigger the corresponding clinical symptoms.

Depending on the relative frequency with which the individual allergen molecules react with the allergic IgE antibodies, a distinction is made between major and minor allergens.

Pollen allergens of different species of the grass family (Poaceae) are classified into groups that are homologous to each other.

In particular, the molecules of group 4 of major allergens have high cross-immunological reactivity with each other, both with mouse monoclonal antibodies and with human IgE antibodies (Fahlbusch et al., 1993 Clin. Exp. Allergy 23: 51-60; Leduc- Brodard et al., 1996, J. Allergy Clin. Immunol. 98: 1065-1072; Su et al., 1996, J. Allergy Clin. Immunol. 97: 210; Fahlbusch et al., 1998, Clin. Exp. Allergy 28: 799-807; Gavrovic-Jankulovic et al.,

30 2000, Invest. Allergol Clin. Immunol 10 (6): 361-367; Stumvoll et al. 2002, Biol. Chem. 383: 1383-1396; Grote et al., 2002, Biol. Chem. 383: 1441-1445; Andersson and Lidholm, 2003, Int. Arch. Allergy Immunol. 130: 87-107; Mari, 2003, Clin. Exp. Allergy, 33 (1): 43-51).

Until now, a complete DNA sequence of no major group 4 allergen is known.

Of the allergens of group 4 of Dactylus glomerata, until now, they have only been obtained by enzymatic degradation and the peptides have been sequenced:

DIYNYMEPYVSK (SEQ ID NO 13),

VDPTDYFGNEQ (SEQ ID NO 14),

ARTAWVDSGAQLGELSY (SEQ ID NO 15),

and GVLFNIQYVNYWFAP (SEQ ID NO 16, Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98: 1065-1072).

40 They have also been obtained by proteolysis and peptides of the group 4 allergens of the Bermuda subtropical grass (Cynodon dactylon) have been sequenced:

KTVKPLYIITP (SEQ ID NO 17),

KQVERDFLTSLTKDIPQLYLKS (SEQ ID NO 18),

TVKPLYIITPITAAMI (SEQ ID NO 19),

LRKYGTAADNVIDAKVVDAQGRLL (SEQ ID NO 20), KWQTVAPALPDPNM (SEQ ID NO 21), VTWIESVPYIPMGDK (SEQ ID NO 22), GTVRDLLXRTSNIKAFGKY (SEQ ID NO 23),

5 TSNIKAFGKYKSDYVLEPIPKKS (SEQ ID NO 24), YRDLDLGVNQVVG (SEQ ID NO 25), SATPPTHRSGVLFNI (SEQ ID NO 26), and AAAALPTQVTRDIYAFMTPYVSKNPRQAYVNYRDLD (SEQ ID NO. 27, Bio.

Research Communication 280: 738-743). 10 For perennial Lolium peptide fragments of group 4 basic allergens were described with the sequences

following: FLEPVLGLIFPAGV (SEQ ID NO 28) and GLIEFPAGV (SEQ ID NO 29, Jaggi et al., 1989, Int. Arch. Allergy Appl. Immunol. 89: 342-348). As the first sequence of a group 4 allergen, it has been resolved by the inventor of the present application for

patent the unpublished sequence of Phl p 4 of Phleum pratense (SEQ ID NO 11) and is described in international application WO 04/000881.

So far nothing is known about the sequences of the major allergens of group 4 cereals (Triceae). Accordingly, the object on which the present invention is based is the preparation of DNA sequences of major allergens of group 4 of cereals, in particular of the Sec c rye allergen (Secale cerale) (SEQ ID NO 1, 3), Hor v 4 of barley (Hordeum vulgare) (SEQ ID NO 5) and Tri to 4 of wheat (Triticum aestivum)

20 (SEQ ID NO 7, 9), as well as the corresponding recombinant DNA molecules on the basis of which the allergens can be expressed as a protein and a significant use can be made accessible as such or in modified form. The sequence of Phl p 4 (SEQ ID NO 11) has been the starting point for the present invention.

Sequence index according to the invention The DNA and protein sequences of mature allergens according to SEQ ID NO 1-10 are preceded by a signal sequence. The coding segment ends with the TGA or TAG stop codons in the DNA sequence.

-

 DNA sequence of Sec c 4. (a) Isoform Sec c 4.01 (SEQ ID NO 1), (b) Isoform Sec c 4.02 (SEQ ID NO 3).

-

 Protein sequences (SEQ ID NO 2, 4) derived from DNA sequences according to SEQ ID NO 1 and 3.

-

 DNA sequence of Hor v 4 (SEQ ID NO 5).

-

 Protein sequence (SEQ ID NO 6) derived from the DNA sequence according to SEQ ID NO 5. 30 - DNA sequence from Tri to 4. (a) Isoform Tri to 4.01 (SEQ ID NO 7), (b) Isoform Tri to 4.02 (SEQ ID NO 9).

-

 Protein sequences (SEQ ID NO 8, 10) derived from DNA sequences according to SEQ ID NO 7 and 9.

-

 Phl p 4 DNA sequence (SEQ ID NO 11) according to SEQ ID NO 5 of WO 04/000881.

-

 Phl p 4 protein sequence (SEQ ID NO 12) according to SEQ ID NO 6 of WO 04/000881. Description of the invention

With the present invention, the DNA sequences of the major allergen of the cereal pollen Sec c 4, Hor v 4 and Tri a 4 according to SEQ ID NO 1, 3, 5, 7, and 9 are now provided for the first time. , the subject of the present invention are DNA molecules with the nucleotide sequence according to

SEQ ID NO 5.

Accordingly, the invention also concerns sequences homologous to the DNA sequence according to the invention or corresponding DNA molecules of the allergens of group 4 of other Poaceae, such as Lolium perennial, Dactylis glomerata, Poa pratensis, Cynodon dactylon and Holcus lanatus , which hybridize under stringent conditions because of the homology in the sequences with the DNA according to the invention, or have a

5 immunological cross-reactivity with respect to the allergens according to the invention.

The invention also includes fragments, novel combinations of partial sequences and point mutations with hypoallergenic effect.

Accordingly, the corresponding partial sequences, a combination of partial sequences or exchange, deletion or addition mutations, which code for a fragment are also subject to the invention.

10 immunomodulator and reagent against T cells of a Poaceae group 4 allergen.

With the knowledge of the DNA sequence of allergens that exist in nature, it is now only possible to prepare these allergens as recombinant proteins, which can be used in the diagnosis and therapy of allergic diseases (Scheiner and Kraft, 1995, Allergy 50: 384-391).

Specific immunotherapy or hyposensitization represents a classic approach to therapeutic treatment

15 Effective of allergies (Fiebig, 1995, Allergo J. 4 (6): 336-339, Bousquet et al., 1998, J. Allergy Clin. Immunol. 102 (4): 558-562). Thus, patients with natural allergens are injected subcutaneously in increasing doses. However, with this method there is a danger of allergic reactions or even anaphylactic shock. To minimize these risks, innovative preparations in the form of allergies are used. These are chemically modified allergen extracts that have a clearly reduced IgE reactivity, although

20 have identical reactivity against T cells compared to the untreated extract (Fiebig, 1995, Allergo J. 4 (7): 377-382).

A broader optimization of recombinant-prepared allergen therapy would be possible. Defined cocktails, where appropriate based on the patient's individual sensitization pattern, of high purity recombinant allergens could replace extracts from natural allergen sources, since

These, apart from the different allergens, contain a large amount of immunogenic but non-allergenic accompanying proteins.

Realistic perspectives, which could lead to safe hyposensitization with expression products, offer targeted mutated recombinant allergens in which IgE epitopes have been specifically removed, without harming the T-cell epitopes essential for therapy (Schramm et al., 1999, J. Immunol. 162: 2406

30 2414).

Another possibility of therapeutically influencing the altered balance of TH cells in allergy sufferers is immunotherapeutic vaccination with DNA. It is an expression-capable DNA treatment that codes for the relevant allergens. The first experimental tests of the allergen-specific influence of the immune response were obtained in rodents by injection of allergen-encoding DNA (Hsu et al.,

35 1996, Nature Medicine 2 (5): 540-544).

Therefore, a DNA molecule described previously or below, or a corresponding recombinant expression vector as a medicament, is also the subject of the present invention.

The corresponding recombinantly prepared proteins can be used for therapy and for in vitro and in vivo diagnosis of pollen allergies.

For the preparation of the recombinant allergen, the cloned nucleic acid is linked to an expression vector and this construct is expressed in an appropriate host organism. After biochemical purification, this recombinant allergen is obtained for the detection of IgE antibodies in established procedures.

Accordingly, a recombinant expression vector containing a DNA molecule described previously or subsequently, functionally linked to a control sequence of the invention is also the subject of the present invention.

Expression and a host organism, transformed with the indicated DNA molecule or the indicated expression vector.

Likewise, the object of the invention is the use of at least one DNA molecule described above or at least one expression vector described above for the preparation of a medicament for immunotherapeutic vaccination with DNA of patients with allergies in whose activation allergens are involved. group 4

50 of Poaceae, preferably Triticeae, in particular Sec c 4, Hor v 4, Tri to 4, and / or for the prevention of such allergies.

As already mentioned, the invention can be used as an essential component in a preparation containing allergens or recombinant nucleic acids for specific immunotherapy. Thus, several possibilities are presented. On the one hand, the unmodified protein in the primary structure can be part of the preparation. On the other hand, by means of the directed deletion of IgE epitopes of the whole molecule or by the preparation of individual fragments encoding T-cell epitopes, a hypoallergenic (allergoid) form is used for therapy according to the invention to avoid unwanted side effects. . Finally, by means of the nucleic acid itself, when it is linked with a eukaryotic expression vector, a preparation that directly modifies the allergic immune state in the therapeutic sense is achieved.

In addition, the present invention relates to polypeptides encoding one or more DNA molecules described above, preferably in their quality as medicaments.

Thus, they are proteins corresponding to an amino acid sequence according to SEQ ID NO 6. In particular it is the mature protein (without the signal sequence part), starting with amino acid 23 (SEQ ID NO 6). In addition, the invention relates to proteins that contain these amino acid sequences or parts of these sequences.

Furthermore, the invention also relates to a process for the preparation of said polypeptides by culturing a host organism and obtaining the corresponding polypeptides from the culture.

Also according to the invention at least one polypeptide described above is used for the preparation of a medicament for the diagnosis and / or treatment of allergies in whose activation allergens of group 4 of Poaceae are involved, preferably Triticeae, in particular Sec c 4, Hor v 4, Tri to 4, as well as for the prevention of such allergies.

To determine the protein and DNA sequences according to the invention, proceed as follows:

Sec c 4 of rye

one.

Starting from the Phl p 4 DNA sequence (SEQ ID NO 12, WO 04/000881) specific primers (Tab. 1) derived from the Phl p 4 sequence were generated. By PCR with primers # 87 and # 83 were obtained five clones of rye pollen DNA. Fragment 1 of the amplified Sec c 4 gene corresponding to these clones encodes a polypeptide corresponding to amino acids 68-401 of Phl p 4 (SEQ ID NO 12).

2.

 With the partial sequence Sec c 4, a search was made in the EST database. However, homologous sequences could not be found in the EST database specialized in rye. Instead, individual, homologous, non-overlapping EST fragments were found in EST databases specializing in barley and wheat. The individual EST fragments reach up to the 5’-UTR segment, others to the 3’-UTR segment (UTR = untranslated segment) of the corresponding genes.

3.

 However, from the EST sequences found in the databases, no complete group 4 gene of wheat or barley can be constructed, since these sequences do not overlap and no homologous group 4 gene is known. , these EST sequences could be classified according to the Phl p 4 sequence (SEQ ID NO 11) and the Sec c 4 fragment obtained in step 1 and served as a standard for the preparation of PCR primers.

Four.

 With the help of primers # 195 and # 189 prepared in this way, three clones could be obtained by PCR. Primer # 195 derived from a barley EST sequence, primer # 189 is a specific Phl p 4 primer and overlaps the Phon p 4 stop codon, as well as the codons of the 10 C-terminal C 4 amino acids of Phl p 4 The fragment 2 of Sec c 4 amplified in this way encodes a polypeptide, starting within the signal sequence and ending with the position corresponding to position 490 of Phl p 4. This polypeptide covers the N-terminus of Sec c 4 .

5th. Three other clones were obtained by PCR with primers # 195 and # 202. Both primers derived from the EST sequences of barley. The amplified Sec c 4 gene 3 encodes the corresponding amino acids starting within the signal sequence and ending at the C terminus of Sec c 4.

The complete sequence of mature Sec c 4 is thus obtained in the defined sequence.

The following two stages 5b and 5c serve to ensure the results obtained in stage 5a:

5b Another clone was obtained by PCR with primers # 195 and # 203. Primer # 195 derived from an EST sequence of barley and primer # 203, from an EST sequence of wheat. The amplified Sec c 4 gene codes for

corresponding amino acids beginning within the signal sequence and ending at the C-terminus of Sec c 4.

The complete sequence of mature Sec c 4 is thus obtained in the defined sequence. 5c. Another clone was obtained by PCR with primers # 195 and # 198. The amplified Sec c 4 gene codes for corresponding amino acids beginning within the signal sequence and ending at the C-terminus of Sec c 4. The complete sequence of mature Sec c 4 is thus obtained in the defined sequence.

Two isoforms Sec c 4.01 and 4.02 were found. Mature allergens begin with amino acid 23 of the sequence according to SEQ ID NO 2, 4 and 6.

Hor v 4 of barley With the help of the Sec c 4 sequences obtained as previously described, they have been found in the EST databases EST fragments homologous to Hordeum vulgare. However, these fragments do not overlap. forming a complete gene. Although according to the EST sequences found, primers could be generated specific to Hor v 4, which were used for amplification of the Hor v 4 gene from genomic DNA.

In total, 15 clones were analyzed. 4 clones were obtained by PCR with primers # 195 and # 198. 4 clones were obtained by PCR with primers # 195 and # 202. 3 clones were obtained by PCR with primers # 194 and # 198. 4 clones were obtained by PCR with primers # 194 and # 202. The sequence of the derived protein begins within the signal sequence of Hor v 4 and reaches the C-terminus

terminal of the protein (from amino acid 23 of SEQ ID NO 6). Tri to 4 wheat With the help of the sequence Sec c 4 obtained as described previously they have been found in the bases

EST data EST fragments homologous to Triticum aestivum. However, these fragments do not overlap forming a complete gene. Although according to the EST sequences found, specific primers of Tri to 4 # 199, # 203, # 204 and # 206 could be generated, which were used for amplification of the Tri to 4 gene from genomic DNA.

In total, 13 clones were analyzed. 4 clones were obtained by PCR with primers # 204 and # 203. 4 clones were obtained by PCR with primers # 204 and # 199. 3 clones were obtained by PCR with primers # 206 and # 203. 4 clones were obtained by PCR with primers # 206 and # 199. The derived protein sequences begin within the signal sequence of Tri to 4 and reach the extreme

C-terminal protein.

Two variants Tri to 4.01 (from amino acid 22 of SEQ ID NO 8) and Tri to 4.02 (from amino acid 22 of SEQ ID NO 10) were found. For the preparation of the recombinant allergens according to the invention DNA sequences were constructed according to

SEQ ID NO 1, 3, 5, 7 and 9 in expression vectors (e.g., pProEx, pSE 380). For N-terminal amino acids

known from protein sequencing, optimized codons of E. coli were employed. After transformation in E. coli, the expression and purification of the recombinant allergen according to the invention by different separation techniques, the protein obtained was subjected to a refolding process.

Both allergens can be used for the highly specific diagnosis of allergies to grass pollen. This diagnosis can be made in vitro by the detection of specific antibodies (IgE, IgG1-4, IgA) and the reaction with effector cells loaded with IgE (eg, blood basophils) or in vivo by reaction reactions in the skin and provocation in the reaction organ.

The reaction of the allergens according to the invention with allergic T lymphocytes to grass pollen can be checked by allergen-specific stimulation of the T lymphocytes for the proliferation and synthesis of cytokine, both with T cells in recently prepared blood lymphocytes and in lines. and established T cell clones reactive against nSec c 4, nHor v 4 or nTri a 4.

By means of site-directed mutagenesis, cysteine coding triplets were modified, so that they encode

10 for other amino acids, preferably serine. Variants were prepared, both in which individual cysteines were replaced and those in which different combinations of 2 cysteine residues or all cysteines were modified. The expressed proteins of these point cysteine mutations have a strongly reduced or non-existent reactivity with allergic IgE antibodies, although they react with the T lymphocytes of these patients.

Accordingly, a DNA molecule described previously or below is also the subject of the present invention, in which by means of site-directed mutagenesis, several or all cysteine residues of the corresponding polypeptide were exchanged for another amino acid.

The immunomodulatory activity of hypoallergenic fragments that correspond to polypeptides with T cell epitopes, as well as that of point hypoallergenic mutations (e.g., cysteine exchanges) can be

20 check by their reaction with the T cells of the allergic to grass pollen.

Such hypoallergenic cysteine fragments or point mutations can be used as preparations for allergic hyposensitization, since they react with the same efficacy with T cells, although due to the reduced or non-existent IgE reactivity they cause few side effects caused by IgE.

If the nucleic acids encoding the hypoallergenic allergen variants according to the invention or the

25 unmodified DNA molecules according to the invention are linked with a human expression vector, these constructs can also be used as preparations for immune therapy (DNA vaccination).

Finally, pharmaceutical preparations containing at least one DNA molecule described above or at least one expression vector described above and, if necessary, other active ingredients and / or adjuvants for immunotherapeutic vaccination with DNA of patients with allergies are subject to the present invention. , in

30 whose activation allergens of group 4 of Poaceae are involved, preferably Triticeae, in particular Sec c 4, Hor v 4, Tri to 4, and / or for the prevention of said allergies.

Another group of pharmaceutical preparations according to the invention, instead of DNA, contains at least one polypeptide described above and is suitable for the diagnosis and / or treatment of the indicated allergies.

Pharmaceutical preparations within the meaning of the present invention contain as active ingredient a polypeptide according to the invention or an expression vector and / or their respective pharmaceutical derivatives, including mixtures thereof in all proportions. Thus, the active ingredients according to the invention can be mixed with at least one solid, liquid and / or semi-liquid excipient or adjuvant and, where appropriate, combined with one or more different active ingredients in a suitable dosage form.

As adjuvants, immunostimulatory DNA or oligonucleotides with CpG motifs are particularly suitable.

These preparations can be used as therapeutic or diagnostic agents in human or veterinary medicine. As excipients organic or inorganic substances suitable for parenteral application and which do not negatively influence the effect of the active ingredient according to the invention come into consideration. For parenteral application, solutions, preferably oily or aqueous solutions, other suspensions, emulsions or implants are used in particular. The active ingredient according to the invention can also be lyophilized and the lyophilisates obtained can be used, for example, for the preparation of injectable preparations. The indicated preparations may be sterilized and / or may contain adjuvants such as preservatives, stabilizers and / or humectants, emulsifiers, salts to influence osmotic pressure, buffer substances and / or various principles.

50 different assets.

Furthermore, by means of the corresponding formulation of the active ingredient according to the invention, deposit preparations can be obtained, for example by adsorption on aluminum hydroxide.

Therefore, the invention also serves to improve in vitro diagnosis within the framework of an identification of allergen components activating the specific patient sensitization spectrum. The invention also serves for the preparation of clearly improved preparations for the specific immunotherapy of allergies to grass pollen.

Table 1 Primers used

a) Sec c 4

Primer No.

SEQ ID NO Sequence

# 0083

30 GGCTCCCGGGGCGAACCAGTAG

# 0087

31 ACCAACGCCTCCCACATCCAGTC

# 0189

32 GATAAGCTTCTCGAGTGATTAGTACTTTTTGATC AGCGGCGGGATGCTC

# 0195

33 GCTCTCGATCGGCTACAATGGCG

# 0198

3. 4 CACGCACTACAAATCTCCATGCAAG

# 0202

35 CATGCTTGATCCTTATTCTACTAGTTGGGC

# 0203

36 TACGCACGATCCTTATTCTACTAGTTGGGC

a) Hor v 4

Primer No.

SEQ ID NO Sequence

# 0194

37 GCCTTGTCCTGCCACCACGCCGCCGCCACC

# 0195

38 GCTCTCGATCGGCTACAATGGCG

# 0198

39 CACGCACTACAAATCTCCATGCAAG

# 0202

40 CATGCTTGATCCTTATTCTACTAGTTGGGC

c) Tri to 4

Primer No.

SEQ ID NO Sequence

# 0199

41 CACGCACTAAATCTCCATGCAAG

# 0203

42 TACGCACGATCCTTATTCTACTAGTTGGGC

# 0204

43 AAGCTCTATCGCCTACAATGGCG

# 0206

44 GGTGCTCCTCTTCTGCGCCTTGTCC

PROT signal sequence

DNA

stop codon

DNA signal sequence

PROT signal sequence

DNA

stop codon

DNA signal sequence

PROT signal sequence

DNA

DNA

undetermined amino acid

DNA DNA

DNA

DNA

DNA DNA

DNA

DNA

DNA

DNA DNA

DNA

DNA

DNA DNA

Claims (10)

1. DNA molecule comprising the nucleotide sequence of a major allergen of cereal pollen according to SEQ ID NO 5.

2.

Recombinant DNA expression vector or a cloning system containing a DNA molecule according to claim 1, functionally linked with an expression control sequence.

3. Method for the preparation of a polypeptide, encoded by a DNA sequence according to claim 1, by culturing a host organism and obtaining the corresponding polypeptide from the culture.

Four.

Polypeptide comprising the amino acid sequence according to SEQ ID NO 6, which codes for a DNA sequence according to claim 1.

5.

Polypeptide comprising the mature allergen of the amino acid sequence according to SEQ ID NO 6.

6.

Polypeptide according to claim 4 or 5 as a medicament.

7.

Pharmaceutical preparation containing at least one polypeptide according to claim 6 for the diagnosis and / or treatment of allergies in whose activation allergens of group 4 of Poaceae are involved.

Use of at least one polypeptide according to claim 6 for the preparation of a medicament for the diagnosis and / or treatment of allergies in whose activation allergens of group 4 of Poaceae and / or for the prevention of said allergies are involved.

9.

DNA molecule according to claim 1 as a medicament.

10.

Recombinant expression vector according to claim 7 as a medicament.

11. Pharmaceutical preparation containing at least one DNA molecule according to claim 9 or at least one expression vector according to claim 10 for immunotherapeutic vaccination with DNA of patients with allergies in whose activation allergens of group 4 of Poaceae are involved and / or for the prevention of such allergies. 12. Use of at least one DNA molecule according to claim 9 or at least one expression vector according to the Claim 10 for the preparation of a medicament for immunotherapeutic vaccination with DNA of patients with allergies in whose activation allergens of group 4 of Poaceae are involved and / or for the prevention of said allergies.